The fitness burden imposed by synthesising quorum sensing signals.

It is now well established that bacterial populations utilize cell-to-cell signaling (quorum-sensing, QS) to control the production of public goods and other co-operative behaviours. Evolutionary theory predicts that both the cost of signal production and the response to signals should incur fitness costs for producing cells. Although costs imposed by the downstream consequences of QS have been shown, the cost of QS signal molecule (QSSM) production and its impact on fitness has not been examined. We measured the fitness cost to cells of synthesising QSSMs by quantifying metabolite levels in the presence of QSSM synthases. We found that: (i) bacteria making certain QSSMs have a growth defect that exerts an evolutionary cost, (ii) production of QSSMs negatively correlates with intracellular concentrations of QSSM precursors, (iii) the production of heterologous QSSMs negatively impacts the production of a native QSSM that shares common substrates, and (iv) supplementation with exogenously added metabolites partially rescued growth defects imposed by QSSM synthesis. These data identify the sources of the fitness costs incurred by QSSM producer cells, and indicate that there may be metabolic trade-offs associated with QS signaling that could exert selection on how signaling evolves.

Impact of an educational intervention upon the hand hygiene compliance of children.

BACKGROUND: Hand hygiene compliance is the single most effective way to reduce healthcare-associated infections. Children are notoriously vulnerable to infection as well as acting as conduits to transmission. Based on these observations, the authors formulated the hypothesis that behavioural change which improved children's hand hygiene compliance would decrease the spread of infectious diseases. AIM: To create an educational intervention to induce long-term behavioural change culminating in increased hand hygiene compliance of children, and thus a decrease in the rate of infections. METHODS: Focus groups conducted during interactive teaching sessions identified what children felt would help them to increase their hand hygiene compliance. This informed the design of an educational device that was subsequently trialled to measure its effectiveness in increasing hand hygiene compliance. Initial developmental stages were conducted in two schools in the East Midlands with study participants aged 5-8 years; the device was subsequently used in a healthcare setting to assess deployment flexibility. FINDINGS: Focus groups indicated that children enjoyed interactive learning, developed knowledge about cross-transmission of infections, and became motivated to encourage others to improve hand hygiene compliance. Microbiological swabbing verified the presence of pathogens on children's hands and environmental surfaces that could serve as reservoirs of infection, and questionnaires indicated an increase in handwashing following the intervention. CONCLUSION: Educational interventions have the potential to increase hand hygiene and reduce the transmission of infections.

Characteristics of a widespread community cluster of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza in Australia.

BACKGROUND: Oseltamivir resistance in A(H1N1)pdm09 influenza is rare, particularly in untreated community cases. Sustained community transmission has not previously been reported. METHODS: Influenza specimens from the Asia-Pacific region were collected through sentinel surveillance, hospital, and general practitioner networks. Clinical and epidemiological information was collected on patients infected with oseltamivir-resistant viruses. RESULTS: Twenty-nine (15%) of 191 A(H1N1)pdm09 viruses collected between May and September 2011 from Hunter New England (HNE), Australia, contained the H275Y neuraminidase substitution responsible for oseltamivir resistance. Only 1 patient had received oseltamivir before specimen collection. The resistant strains were genetically very closely related, suggesting the spread of a single variant. Ninety percent of cases lived within 50 kilometers. Three genetically similar oseltamivir-resistant variants were detected outside of HNE, including 1 strain from Perth, approximately 4000 kilometers away. Computational analysis predicted that neuraminidase substitutions V241I, N369K, and N386S in these viruses may offset the destabilizing effect of the H275Y substitution. CONCLUSIONS: This cluster represents the first widespread community transmission of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza. These cases and data on potential permissive mutations suggest that currently circulating A(H1N1)pdm09 viruses retain viral fitness in the presence of the H275Y mutation and that widespread emergence of oseltamivir-resistant strains may now be more likely.

Quantitative liquid chromatography-tandem mass spectrometry profiling of activated methyl cycle metabolites involved in LuxS-dependent quorum sensing in Escherichia coli.

A rapid, selective, and sensitive liquid chromatography-tandem mass spectrometry assay has been developed and validated for the simultaneous quantification of the metabolites and precursors of the activated methyl cycle, reported in preliminary form by Heurlier et al. (2009) [43]. Analytes were extracted from Escherichia coli MG1655 and chemically derivatized as N(O,S)-iso-butyloxycarbonyl iso-butyl esters using iso-butyl chloroformate in an aqueous iso-butanol/pyridine environment. S-Adenosylmethionine, S-adenosylhomocysteine, S-ribosylhomocysteine, homocysteine, methionine, cystathionine, cysteine, and homoserine were quantified by liquid chromatography-positive ion tandem electrospray ionization mass spectrometry. Internal standards were isotopically labeled [(13)CD(3)]methionine and S-adenosylcysteine. Linearity of the assay was established up to a concentration of 700 microg/g cell dry weight for each analyte. The validated assay was used to quantitatively profile the intracellular activated methyl cycle metabolites as a function of growth in E. coli MG1655 and its derivative Deltapfs and DeltaluxS mutants to determine the metabolic consequences of a disruption to the activated methyl cycle and, hence, LuxS-dependent quorum sensing.

The evolution of nuclear receptors: evidence from the coral Acropora.

We have amplified and sequenced PCR products derived from 10 nuclear receptor (NR) genes from the anthozoan cnidarian Acropora millepora, including five products corresponding to genes not previously reported from the phylum Cnidaria. cDNAs corresponding to seven of these products were sequenced and at least three encode full-length proteins, increasing the number of complete cnidarian NR coding sequences from one to four. All clear orthologs of Acropora NRs either lack an activation domain or lack a known ligand, consistent with the idea that the ancestral nuclear receptor was without a ligand. Phylogenetic analyses indicate that most, and possibly all, presently identified cnidarian NRs are members of NR subfamily 2, suggesting that the common ancestor of all known nuclear receptors most resembled members of this subfamily.

Genetic dissection of the outer membrane secretin PulD: are there distinct domains for multimerization and secretion specificity?

Linker and deletion mutagenesis and gene fusions were used to probe the possible domain structure of the dodecameric outer membrane secretin PulD from the pullulanase secretion pathway of Klebsiella oxytoca. Insertions of 24 amino acids close to or within strongly predicted and highly conserved amphipathic beta strands in the C-terminal half of the polypeptide (the beta domain) abolished sodium dodecyl sulfate (SDS)-resistant multimer formation that is characteristic of this protein, whereas insertions elsewhere generally had less dramatic effects on multimer formation. However, the beta domain alone did not form SDS-resistant multimers unless part of the N-terminal region of the protein (the N domain) was produced in trans. All of the insertions except one, close to the C terminus of the protein, abolished function. The N domain alone was highly unstable and did not form SDS-resistant multimers even when the beta domain was present in trans. We conclude that the beta domain is a major determinant of multimer stability and that the N domain contributes to multimer formation. The entire or part of the N domain of PulD could be replaced by the corresponding region of the OutD secretin from the pectate lyase secretion pathway of Erwinia chrysanthemi without abolishing pullulanase secretion. This suggests that the N domain of PulD is not involved in substrate recognition, contrary to the role proposed for the N domain of OutD, which binds specifically to pectate lyase secreted by E. chrysanthemi (V. E. Shevchik, J. Robert-Badouy, and G. Condemine, EMBO J. 16:3007-3016, 1997).

In vitro biosynthesis of the Pseudomonas aeruginosa quorum-sensing signal molecule N-butanoyl-L-homoserine lactone.

In Pseudomonas aeruginosa, synthesis of the quorum-sensing signal molecules N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL) requires the Luxl homologue Rhll(Vsml). By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified Rhll can catalyse the biosynthesis of BHL and HHL using either S-adenosylmethionine (SAM) or homoserine lactone (HSL) but not homoserine as the source of the homoserine lactone moiety. As we were unable to detect homoserine lactone in cytoplasmic extracts of Escherichia coli, we conclude that SAM is the natural substrate for Rhll-directed N-acylhomoserine lactone (AHL) biosynthesis. The N-acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n-butanoyl-CoA or n-hexanoyl-CoA). The specificity of Rhll for charged CoA derivatives is demonstrated as Rhll was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n-octanoyl-CoA, n-decanoyl-CoA, DL-beta-hydroxybutanoyl-CoA or crotonoyl-CoA. Rhll was also unable to use N-acetyl-S-3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA. Furthermore, the Rhll-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.

Analysis of the carbapenem gene cluster of Erwinia carotovora: definition of the antibiotic biosynthetic genes and evidence for a novel beta-lactam resistance mechanism.

Members of two genera of Gram-negative bacteria, Serratia and Erwinia, produce a beta-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid. We have reported previously the cloning and sequencing of the genes responsible for production of this carbapenem in Erwinia carotovora. These genes are organized as an operon, carA--H, and are controlled by a LuxR-type transcriptional activator, encoded by the linked carR gene. We report in this paper the genetic dissection of this putative operon to determine the function of each of the genes. We demonstrate by mutational analysis that the products of the first five genes of the operon are involved in the synthesis of the carbapenem molecule. Three of these, carABC, are absolutely required. In addition, we provide evidence for the existence of a novel carbapenem resistance mechanism, encoded by the CarF and carG genes. Both products of these overlapping and potentially translationally coupled genes have functional, N-terminal signal peptides. Removal of these genes from the Erwinia chromosome results in a carbapenem-sensitive phenotype. We assume that these novel beta-lactam resistance genes have evolved in concert with the biosynthetic genes to ensure 'self-resistance' in the Erwinia carbapenem producer.

Recent progress and future directions in studies of the main terminal branch of the general secretory pathway in Gram-negative bacteria--a review.

The main terminal branch (MTB) of the general secretory pathway is used by a wide variety of Gram- bacteria to transport exoproteins from the periplasm to the outside milieu. Recent work has led to the identification of the function of two of its 14 (or more) components: an enzyme with type-IV prepilin peptidase activity and a chaperone-like protein required for the insertion of another of the MTB components into the outer membrane. Despite these important discoveries, little tangible progress has been made towards identifying MTB components that determine secretion specificity (presumably by binding to cognate exoproteins) or which form the putative channel through which exoproteins are transported across the outer membrane. However, the idea that the single integral outer membrane component of the MTB could line the wall of this channel, and the intriguing possibility that other components of the MTB form a rudimentary type-IV pilus-like structure that might span the periplasm both deserve more careful examination. Although Escherichia coli K-12 does not normally secrete exoproteins, its chromosome contains an apparently complete set of genes coding for MTB components. At least two of these genes code for functional proteins, but the operon in which twelve of the genes are located does not appear to be expressed. We are currently searching for conditions which allow these genes to be expressed with the eventual aim of identifying the protein(s) that E. coli K-12 can secrete.

The C-terminal domain of the secretin PulD contains the binding site for its cognate chaperone, PulS, and confers PulS dependence on pIVf1 function.

Related outer membrane proteins, termed secretins, participate in the secretion of macromolecules across the outer membrane of many Gram-negative bacteria. In the pullulanase-secretion system, PulS, an outer membrane-associated lipoprotein, is required both for the integrity and the proper outer membrane localization of the PulD secretin. Here we show that the PulS-binding site is located within the C-terminal 65 residues of PulD. Addition of this domain to the filamentous phage secretin, pIV, or to the unrelated maltose-binding protein rendered both proteins dependent on PulS for stability. A chimeric protein composed of bacteriophage f1 pIV and the C-terminal domain of PuID required properly localized PulS to support phage assembly. An in vivo complex formed between the pIV-PulD65 chimera and PulS was detected by co-immunoprecipitation and by affinity chromatography.

Pullulanase: model protein substrate for the general secretory pathway of gram-negative bacteria.

Pullulanase of Klebsiella oxytoca is one of a wide variety of extracellular proteins that are secreted by Gram-negative bacteria by the complex main terminal branch (MTB) of the general secretory pathway. The roles of some of the 14 components of the MTB are now becoming clear. In this review it is proposed that most of these proteins form a complex, the secretion, that spans the cell envelope to control the opening and closing of channel in the outer membrane. Progress toward the goal of testing this model is reviewed.

Reproducibility of brachial ultrasonography and flow-mediated dilatation (FMD) for assessing endothelial function.

BACKGROUND: High-resolution brachial artery ultrasonography is used to study vasodilator response induced by physiologic reactive hyperaemia. We examined the reproducibility of measuring flow-mediated dilatation (FMD) on two occasions. AIMS: To determine the degree of variability of this technique in our vascular laboratory for the design of clinical research studies. METHODS: Nineteen subjects were studied on two separate occasions using an Acuson 128 ultrasound device and a 7.0 MHz linear array transducer. Reactive hyperaemia was induced in the brachial artery by inflation and release of a blood pressure cuff. Nitrate-induced dilatation was assessed in 11 of the 19 subjects. Measurements were made by two observers blinded to subject details. RESULTS: The 11 subjects given sublingual GTN during the first ultrasound study had a mean nitrate-induced dilatation of 20.7% (sd 9.6). The mean vessel diameter of 3.78 mm (sd 0.7) at rest and 3.89 mm (sd 0.7) during reactive hyperaemia yielded a mean FMD of only 3.0% (sd 2.7). The mean difference in FMD within-observers was 0.13% (sd 2.07), between-observers 0.06% (sd 2.17) and between-studies was 0.57% (sd 6.83). CONCLUSIONS: The reproducibility of FMD measured by brachial artery ultrasound was poor and likely to render the measurements inaccurate for clinical research in our hands. Between-study variation contributed the largest proportion of total study variability. We suggest that investigators using this technique conduct their own careful reproducibility studies in order to avoid the misinterpretation of 'negative' studies.

The secretin-specific, chaperone-like protein of the general secretory pathway: separation of proteolytic protection and piloting functions.

The chaperone-like protein of the main terminal branch of the general secretory pathway from Klebsiella oxytoca, the outer membrane lipoprotein PulS, protects the multimeric secretin PulD from degradation and promotes its correct localization to the outer membrane. To determine whether these are separable functions, or whether resistance to proteolysis results simply from correct localization of PulD, we replaced the lipoprotein-type signal peptide of PulS by the signal peptide of periplasmic maltose-binding protein. The resulting periplasmic PulS retained its ability to protect PulD, but not its ability to localize PulD to the outer membrane and to function in pullulanase secretion. Periplasmic PulS competed with wild-type PulS to prevent pullulanase secretion, presumably again by causing mislocalization of PulD. A hybrid protein comprising the mature part of PulS fused to the C-terminus of full-length maltose-binding protein (MalE-PulS) had similar properties to the periplasmic PulS protein. Moreover, MalE-PulS was shown to associate with PulD by amylose-affinity chromatography. The MalE-PulS hybrid was rendered completely functional (i.e. it restored pullulanase secretion in a pulS mutant) by replacing its signal peptide with a lipoprotein-type signal peptide. However, this fattyacylated hybrid protein was only functional if it also carried a lipoprotein sorting signal that targeted it to the outer membrane. Thus, the two functions of PulS are separate and fully dissociable. Incorrect localization, rather than proteolysis, of PulD in the absence of PulS was shown to be the factor that causes high-level induction of the phage shock response. The Erwinia chrysanthemi PulS homologue, OutS, can substitute for PulS, and PulS can protect the secretin OutD from proteolysis in Escherichia coli, indicating the possible existence of a family of PulS-like chaperone proteins.

Independent interaction of the acyltransferase HlyC with two maturation domains of the Escherichia coli toxin HlyA.

The apparently unique fatty acylation mechanism that underlies activation (maturation) of Escherichia coli haemolysin and related toxins is further clarified by investigation of the interaction of protoxin with the specific acyltransferase HlyC. Using deleted protoxin variants and protoxin peptides as substrates in an in vitro maturation reaction dependent upon HlyC and acyl-acyl carrier protein, two independent HlyC recognition domains were identified on the 1024-residue protoxin, proA, and they were shown to span the two target lysine residues K564 (KI) and K690 (KII) that are fatty acylated. Each domain required 15-30 amino acids for basal recognition and 50-80 amino acids for wild-type acylation. The two domains (FAI and FAII) competed with each other in cis and in trans for HlyC. The affinity of FAI for HlyC is approximately four times greater than that of FAII resulting in an overall 80% acylation at KI and 20% acylation at KII in both whole toxin and peptide derivatives. No other proA sequences were required for toxin maturation, and excess Ca2+ prevented acylation of both lysines. The lack of primary sequence identity between FAI and FAII domains in proA and among corresponding sites on related protoxins currently precludes an explanation of the basis of HlyC recognition by proA.

Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein.

Only one of the characterized components of the main terminal branch of the general secretory pathway (GSP) in Gram-negative bacteria, GspD, is an integral outer membrane protein that could conceivably form a channel to permit protein transport across this membrane. PulD, a member of the GspD protein family required for pullulanase secretion by Klebsiella oxytoca, is shown here to form outer membrane-associated complexes which are not readily dissociated by SDS treatment. The outer membrane association of PulD is absolutely dependent on another component of the GSP, the outer membrane-anchored lipoprotein PulS. Furthermore, the absence of PulS resulted in limited proteolysis of PulD and caused induction of the so-called phage shock response, as measured by increased expression of the pspA gene. We propose that PulS may be the first member of a new family of periplasmic chaperones that are specifically required for the insertion of a group of outer membrane proteins into this membrane. PulS is only the second component of the main terminal branch of the GSP for which a precise function can be proposed.

Vibrio spp. secrete proaerolysin as a folded dimer without the need for disulphide bond formation.

Proaerolysin is an extracellular dimeric protein that is secreted across the inner and outer membranes of Aeromonas spp. in separate steps. To investigate the role of protein folding in the second step, one or more cysteine residues were introduced and the mutant proaerolysins were expressed in Aeromonas hydrophila and Aeromonas salmonicida, as well as Vibrio cholerae. Replacing Met-41 with Cys resulted in expression of a protein that could form a dimer in which the monomers were linked together by a disulphide bridge. A double mutant was also made, in which Gly-202 and Ile-445 were replaced with cysteine in order to allow the formation of an intrachain disulphide bridge when the molecule was correctly folded. The M41C covalent dimer and G202C/I445C proaerolysin with the new intrachain bridge were both easily detected inside the bacteria, and they later appeared in the culture supernatants. Small amounts of incorrectly folded proaerolysin were also observed in the cells, but they were not secreted. We observed in the cells, but they were not secreted. We conclude that proaerolysin folds and dimerizes before being released from the cell, and that correct folding is a requirement for secretion to occur. The proton ionophore CCCP reduced release of the folded proteins. Unoxidized protein was secreted by cells grown in beta-mercaptoethanol and by a dsbA mutant of V. cholerae, indicating that disulphide bond formation may not be essential for release.

The C-terminal peptide produced upon proteolytic activation of the cytolytic toxin aerolysin is not involved in channel formation.

The channel-forming toxin aerolysin is secreted by Aeromonas hydrophila as a protoxin that can be activated by nicking with endoproteinase Lys-C after Lys-427 near the C terminus of the protein. The fate of the 43-amino acid peptide distal to the activation site was investigated. A cysteine was introduced into the C-terminal region by replacing Ile-445, and another replaced Gly-202, which is on the proximal side of the activation site. In a double mutant, the two new cysteines were close enough in the folded molecule to form an intrachain 202-445 disulfide bond. Tryptophan fluorescence measurements on wild type and the 2 single cysteine mutants indicated that activation results in exposure of at least 1 tryptophan residue, leading to the conclusion that the peptide moves with respect to the protein when it is produced. This was supported by the observation that upon activation there was a decrease in energy transfer between a tryptophan in the bulk of the protein and a probe attached to Cys-445. The peptide could be separated from active toxin by several methods, indicating that it leaves the protein when it is produced, and that it plays no further role in the process of channel formation.

Analysis by pulsed-field gel electrophoresis of insertion mutations in the transferrin-binding system of Haemophilus influenzae type b.

A mutagenesis system involving the insertion of a non-transposable antibiotic resistance gene cassette was used to generate stable mutations in the chromosome of Haemophilus influenzae type b strain Eagan. The mutations generated were shown by pulsed-field gel electrophoresis (PFGE) to have unique SmaI fingerprint patterns and to be located randomly on the chromosome. Of 700 insertion mutants screened, 29 had stable insertions resulting in constitutive expression of transferrin-binding proteins (TBPs). The high proportion of such mutants indicated that numerous regulatory loci could influence the expression of this phenotype. Five such regulatory mutations were analysed in detail by PFGE and DNA hybridisation and were shown to be located at five different chromosomal loci, although three of the five loci were located on the same 330-kb SmaI fragment of the wild-type strain Eagan chromosome. This fragment also contains several important virulence determinants, including the capb locus, and one of the five constitutive mutants had concomitantly lost the ability to synthesise a type-b capsule. No DNA homology was demonstrated between H. influenzae chromosomal fragments separated by PFGE and DNA probes for the TBPs from Neisseria meningitidis, but the possibility of shared regulatory mechanisms controlling the expression of TBPs in these two species remains to be investigated.

Pore formation in artificial membranes by the secreted hemolysins of Proteus vulgaris and Morganella morganii.

Lipid-bilayer experiments were performed with the related hemolysins from Proteus vulgaris and Morganella morganii (HlyA). The addition of the toxins to the aqueous phase bathing lipid-bilayer membranes composed of different lipids resulted in the formation of transient ion-permeable channels. Membranes formed of pure lipids were rather inactive targets for the hemolysins as compared with lipid mixtures such as asolectin. The channels had several different substrates. The major open state had single-channel conductances of 500 pS in 0.15 M KCl at small transmembrane voltages. Experiments with different salts suggested that the hemolysin-induced channels of P. vulgaris and M. morganii were exclusively cation selective at neutral pH, caused by negative charges localized at the channel mouth. The mobility sequence of the cations within the channels was similar if not identical to their mobility sequence in the aqueous phase. The single-channel data were consistent with wide, water-filled channels with estimated minimal diameters of about 1 nm since the large organic cation Tris+ can permeate the channels without any detectable interaction with its interior. Pore-forming properties of these hemolysins were compared with those of HlyA of Escherichia coli. All these toxins share common features, oligomerize probably to form pores in lipid-bilayer membranes and form channels with similar properties which suggests that their structures are more or less identical.

Transferrin-binding ability of invasive and commensal isolates of Haemophilus spp.

Haemophilus influenzae type b expresses an inducible siderophore-independent iron-acquisition system that depends on a direct interaction between human transferrin and specific iron-regulated transferrin-binding outer-membrane proteins. To evaluate the importance of this iron-acquisition system amongst haemophili, 156 isolates of Haemophilus spp. (78 commensal isolates and 78 isolates from invasive infections) were examined for their ability to bind transferrin. Of the 78 invasive isolates, all of which were H. influenzae type b, 71 (91%) were capable of binding transferrin, with 57 (73%) binding transferrin constitutively (i.e., even when grown in an iron-sufficient medium). In contrast, only 11 (14%) of the commensal isolates bound transferrin constitutively, with a further 16 (21%) binding transferrin only after growth in an iron-deficient medium. Of the 27 commensal strains that were capable of binding transferrin, 12 were H. parainfluenzae biotype III, 14 were non-typable H. influenzae, and one was H. parahaemolyticus. None of the H. influenzae type b invasive or commensal isolates showed evidence of siderophore production, but 50 (66%) of the remaining 76 commensal isolates appeared to produce an iron chelator. Thus, while not a universal characteristic, detectable transferrin-binding was associated strongly with H. influenzae type b isolates from invasive infections, and was also recognised for the first time in isolates of H. parainfluenzae and H. parahaemolyticus.